Illuminating cellular and extracellular vesicle-mediated communication via a split-Nanoluc reporter in vitro and in vivo

Summary Tools to effectively demonstrate and quantify functional delivery in cellular communication have been lacking. This study reports the use of a fluorescently labeled split Nanoluc reporter system to demonstrate and quantify functional transfer between cells in vitro and in a subcutaneous tumor mouse model. Our construct allows monitoring of direct, indirect, and specifically extracellular vesicle-mediated functional communication.


INTRODUCTION
Intercellular communication is an integral part of maintaining homeostasis in multicellular organisms, with dysregulation playing pivotal roles in tumorigenesis, 1 aging, 2 and infectious disease. 3 Cellular communication is generally separated into direct, contact-dependent communication 4 and indirect communication, which is based on the exchange of soluble factors and extracellular vesicles. 5 Extracellular vesicles, membrane encapsulated particles released from cells via outward budding of the cellular plasma membrane or via fusion of endosomal-derived multivesicular bodies with the plasma membrane, have gathered special interest due to increasing evidence that their contents are selectively packaged and released, and their effects can be exerted far from the cell of origin. 6 There are many tools to study the effects of cellular communication in vitro and in vivo, but those able to do so in real time are scarce. 7 Until now, much work has been limited to monitoring internalization as a means of evaluating functional uptake or analyzing indirect downstream targets. 7 Functional transfer of RNAs via extracellular vesicles (EVs) has been demonstrated through CRISPR-Cas9-based reporter systems, and these tools can be valuable on a single-cell level. 8 However, these are on-off systems where the readout does not equate to the amount of functionally delivered cargo, and translation into in vivo models is complex. 9 Quantification through luminescence is possible when using the commercially available reporter systems (Promega HI, Lgbit); however, these lack fluorescence, 10 making tracking of uptake difficult.
We set out to create a system that would allow for monitoring and quantification of functional protein exchange between cells for direct and indirect cell-to-cell interaction, including EV-mediated communication.

RESULTS
We created two proteins, N65 and 66C, which incorporate a previously developed split Nanoluc protein. 11 These two equivalently MOTIVATION In tracking protein-mediated cellular communication, few tools distinguish between proteins that escape the endosome and are functional within the cytosol and proteins that are degraded by the lysosome or released back into the extracellular space. We designed a tool that signals functional protein delivery and can be used to study cell-to-cell and extracellular vesicle-mediated communication in vitro and in vivo. Report ll OPEN ACCESS sized fragments (N65 is 10.3 kDa and 66C is 13.5 kDa) spontaneously reform when in close proximity to one another. Nanoluc has no innate luminescence but catalyzes a bright stable luminescent signal when incubated with the substrate furimazine (FMZ). 12 We generated fusion proteins of these Nanoluc ''halves'': N65, with fluorescent protein mTurquoise-2 13 and a human influenza hemagglutinin (HA) tag 14 on the N terminus, and 66C, with mScarlet-I 15 and a FLAG tag 16 on the C terminus ( Figures 1A-1D). Transfection of HEK cells with both constructs showed a strong luminescent signal in both the cells and the media when adding FMZ 48 h after transfection ( Figure 1E). There was a strong correlation between the number of transfected cells and the luminescent signal in the cells and media, with p < 0.0001 and R 2 > 0.9 for both ( Figure 1F). We evaluated functional delivery of proteins via direct and indirect culturing methods ( Figure 1G). Direct coculture of HeLa cells led to a strong luminescent signal in the cells and media after 7 days ( Figure 1H). Similarly, we could detect luminescence in cells and media after direct co-culture of breast cancer cell line MDA-MB-231 ( Figure S1A) and immortalized human astrocytes ( Figure S1B). Functional exchange of proteins was also observed in direct co-culture of two different cell types: human-derived glioma cell line U87 and immortalized human astrocytes, although the signal could only be detected in the cells and not in the media ( Figure 1I). The signal strength varied between experiments and was highly dependent on the number of cells fully transduced or the transfected level of transduction per cell as determined by fluorescent signal, total number of cells (as illustrated in Figure 1F), confluency, and length of co-culture (data not shown).
To assess if our tool was able to visualize indirect cellular communication, we performed indirect co-cultures with transduced U87 cells and immortalized astrocytes. We detected a luminescent signal in the cells and in the media ( Figures 1J and  1K). This could indicate either free release and uptake of proteins or EV-mediated transfer of proteins. To evaluate the usefulness of our construct as an EV reporter, we transfected HEK cells with both constructs. Transfection agent was removed after 18 h, and cells were replated in fresh media. Media were collected after 72 h and run through a size-exclusion chromatographer (SEC). Most of the luminescent signal was observed in the protein fractions, while some signal could be observed in the early, EV-associated, fractions (7-11) ( Figure S1C). Previously, we demonstrated that SEC successfully separates and isolates EVs from free proteins. 17 EV-specific protein CD81 18 could be detected in the EV fractions but not in the protein fractions (Figure S2A). To demonstrate that there was no contamination from free proteins in the EV fractions, we treated both EV and protein-associated fractions with proteinase K ( Figure S2B). Proteinase K is a strong proteolytic enzyme that efficiently degrades proteins upon incubation. 19 EVs and proteins were collected from HEK293T cells transfected with both 66c and N65 constructs with SEC, and input was normalized based on fluorescence. The luminescent signal was similar between samples before adding proteinase K, while luminescence significantly decreased after adding proteinase K to the protein fractions. The signal in the EV fraction did not change, indicating that the proteins are indeed protected by the EV membrane and that there is little contamination of free proteins in the EV fractions ( Figure S2B).
After stable transduction HeLa cells, mTurquoise2 and mScarlet-I fluorescence could be detected in the respective EV fractions (Figures 2A, 2B, S2C, and S2D). Next, concentrated EVs were added to HeLa cells transduced with the N65 or the 66C construct. Luminescence was measured after 48 h and could be detected in the recipient cells and in the media ( Figures 2C and 2D). Repeated measurements from the moment of incubation with EVs and FMZ (t = 0), showed that the luminescence increased over time ( Figures 2E and 2F). Finally, we compared the luminescent signal between delivery via EVs and delivery via free proteins. EVs and proteins were isolated from transfected N65 cells via SE, and the concentration of N65 protein was normalized based on fluorescence. Equal amounts were then added to transduced 66C recipient cells. Luminescence was measured after 72 h. With similar protein input, luminescent signal above baseline could be detected in 66C cells receiving N65 EVs and N65 free protein and in the media of cells that received N65 EVs ( Figure S2E). A higher luminescent signal was detected when EVs were added compared with free protein.
To further demonstrate versatility of this tool, we fused a nuclear localization signal (NLS) 20 to the N65 construct (Figure 2G). The NLS causes accumulation of the protein in the cell nucleus, decreasing release into the cytosol and extracellular  Figure 2H). Co-culturing of NLS-N65 transduced HEK cells demonstrated retention of the NLS-fused protein in the nucleus, prohibiting functional uptake in the other cells (Figure 2I). Luminescence was measured in the cell pellet and media 7 days after the start of co-culture. As expected, there was no significant signal in cells and media when co-culturing NLS-N65 with N65 or NLS-N65 HEK cells. A significant increase in luminescence was found when co-culturing NLS-N65 with normal 66C HEK cells but not when co-cultured with NLS-66C. When both N65 and 66C proteins are fused with NLS, they do not leave the nucleus, and thus functional transfer to other cells is inhibited. In this experiment, some increase in luminescence was observed when co-culturing NLS-N65 and NLS-66C compared with control, indicating some leakage of proteins out of the nucleus, but this increase was not significant and not observed in the media ( Figure 2I). Direct co-culture of two NLS constructs did not lead to significant increase in luminescent signal, indicating successful retention of the protein to the nucleus ( Figure 2I). Finally, cellular communication could be detected in vivo. Breast cancer cells MDA-MB-231 were transduced with either the N65 construct or the 66C construct and grown for 7 days. Athymic nude mice were subcutaneously injected with MDA-MB-231-N65 or MDA-MB-231-66C cells or with a mixture of both cell lines. Rapid tumor formation ensued. To visualize functional exchange of proteins, we injected fluorofurimazine (FFz), optimized for in vivo applications of Nanoluc, 21 intraperitoneally in mice weekly and measured in vivo bioluminescence (Figure 2J). We were able to detect a strong luminescent signal in the tumors consisting of both cell lines compared with controls with only one cell line, which increased over time (Figures 2K  and 2L). This indicates that this construct can be utilized to study cellular communication in vivo.

DISCUSSION
In this report, we demonstrate an assay to measure functional delivery of proteins for direct, indirect, and EV-mediated communication. We show the functionality and adaptability of these assays in vitro and in vivo.
Many assays have been developed to measure (EV-mediated) delivery, all with various qualities and shortcomings. Recently a model utilizing the LgBit and HiBit tags was developed, following a similar concept to show functional delivery of proteins. 22 Specifically designed to demonstrate functional delivery via EVs, delivery could be detected via luminescence in real time. As LgBit and HiBit weakly associate, the efficacy of Nanoluc formation depends on the interaction of the target proteins to which they are fused. 12 The rate of Nanoluc formation was too low to be detected, and only after addition of a fusogenic protein could the signal be detected above background. 22 Toribio et al. attempted to create a similar assay using a split EGFP luciferase, with onehalf fused to CD9 and the other half freely expressed within recipient cells, but this did not provide a fluorescent or luminescent signal upon EV-mediated delivery. 23 Only when EVs carried the full reconstituted dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate could uptake be detected. While it can show live uptake and is quantifiable, this assay is not able to distinguish between uptake and actual functional delivery. 23 Similarly, another study was able detect GFP fused to CD63 in donor cells incubated with GFP-CD63 EVs, showing uptake but not specifying functional delivery. 24 Functional delivery of RNAs has been demonstrated by a study utilizing the CRISPR-Cas9 system. 8 Cells were transduced to express mCherry, with EGFP flanked by a stop codon. Upon functional delivery of a single guide RNA, CRISPR-Cas9 removes one or two nucleotides in the linker, creating a frameshift to bypass the stop codons and transcribe the EGFP. Functionality was shown in co-culture and EV-based assays, which demonstrated low efficiency: on average, 0.07% of cells expressed GFP after 5 days of incubation. 8 Our construct offers various advantages over currently available protein-based assays. First, we demonstrate the versatility of this construct, showing its use in direct, indirect, and EVbased cellular communication. As two intact proteins are Report ll OPEN ACCESS required to create the luminescent protein, this assay distinguishes uptake from functional delivery. Furthermore, we show that this construct works both ways, with both halves being suitable to be either donor or receiver, and that the construct can be adapted to study specific aspects of cellular communication without a significant decrease in functionality. This construct uniquely allows monitoring of cellular communication in an in vivo mouse model, with the signal being strong enough to be detected with IVIS.
Overall, our reporter system provides a tool that allows for detection and relative quantification of direct, indirect, and specifically indirect EV-mediated cellular interaction.
We believe that this tool will facilitate researchers to evaluate and improve cellular communication and functional EV-mediated delivery and further aid our understanding of cellular communication in vitro and in vivo.

Limitations of the study
One of the limits of this construct is the difficulty in quantifying the signal. Strength of signal depends on many variables, such as strength of transduction or transfection, number of cells, EVs released, amount of protein packed per EV, and many others. Another limitation is that the construct does not differentiate between methods of delivery; the luminescent signal demonstrates that functional delivery has occurred, not whether this has been through proteins, EVs, or other forms of cellular communication. By carefully controlling experiments, this construct can be used to assess individual forms of cellular communication, but contamination cannot be ruled out based on this construct alone. Furthermore, luminescence does tend to vary between experiments. Comparing functional uptake between two different experiments is therefore difficult, and caution needs to be taken when extrapolating results from single experiment. While other constructs have similar issues, the CRISPR-Cas9 method does allow for better quantifiable response. 8 Functionality of the Nanoluc is, however, not dependent on post-translational modification, as are GFP-based reporter systems, 25 allowing for it to function at lower expression levels with less influence of other cellular processes. 26

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:
Animal studies Ten week old, female athymic nude mice (nu/nu) were obtained from Charles River Laboratories. Per mouse, 1 3

Cloning
Sequences for N65 and 66C were taken from Zhao et al. 11 Sequences for mTurquoise2 and mScarlet-I were added to both, respectively, and ordered from Integrated DNA Technologies (IDT) as a gBlock and cloned into lentiviral backbone (Supplemental Information). We also added c-myc NLS sequence to localize expression to the nucleus, which was also ordered as a gBlock (Supplemental Information). Cloning was performed using Gibson Assembly Cloning kit from New England Biolabs (NEB). Plasmids were transformed in One ShotTOP10 bacterial cells from ThermoFisher Scientific. Subsequent MaxiPrep kits (ThermoFisher Scientific) were used to isolate DNA and samples were then sequenced by the MGH sequencing core.

Western Blot
Cells were trypsinized and lysed with RIPA buffer (Abcam, Cambridge, MA, USA) with a cocktail of Protease Inhibitors (Roche, Mannheim, Germany) and centrifuged for 10 min at 12,000 3 g at 4 C. Protein concentration was quantified with the Pierce BCA Protein Assay kit (Thermo Scientific). 2mg of protein was denatured at 95 C for 5 min. Samples were run on 4-12% NuPAGE Bis-Tris Gel (ThermoFisher Scientific) and transferred to a nitrocellulose membrane (Bio-Rad). After blocking with Tris-Buffered Saline (TBS) with 0.05% Tween 20 (TBS-T) and 5% milk for 1 h, the membrane was incubated with the primary antibody in TBS-T with 3% milk overnight at 4 C. The membrane was washed with TBS-T and incubated with the secondary antibody for 1 h at room temperature. Primary antibodies were: Anti-HA tag, (mouse mAB, 18181, abcam), Anti-Flag (mouse mAB, F3165, Sigma-Aldrich), all 1:100 dilutions. Secondary was ECL Anti-mouse IgG (Thermo Fisher) (1:1000).

Transfection
To introduce plasmids into HEK293T cells, 1 mg/mL polyethylenimine (PEI) (Polyscience, Warrington, PA USA) was added to the media. DNA (4 mg) was mixed with 250 mL Opti-MEMâ (ThermoFisher), and 50 mL PEI was mixed with 200 mL Opti-MEM separately. They were each incubated at RT for 5 min and then combined to a final volume of 500 mL. After the transfection agent was incubated at RT for 20 min, it was dropwise added to the cells and incubated for 18 h after which cells were trypsinized and washed with PBS before replating in fresh media.